Epstein-Barr virus (EBV) is an important cause of human cancers world-wide, including both B cell and epithelial cell malignancies. Although the role of EBV-encoded latency proteins in driving these cancers is well accepted, the importance of lytic viral proteins remains relatively controversial. Nevertheless, lytic infection in a subset of EBV-infected cells in vivo may be required for efficient EBV-induced lymphoma formation. We previously showed that a lytic-defective EBV mutant (missing the BZLF1 (Z) immediate-early (IE) gene) is impaired for the ability to form lymphomas in a humanized mouse model that allows horizontal virus transmission. In addition, we and others have identified growth factors and immunosuppressive factors released from lytically infected B cells that likely enhance the growth and survival of nearby latently infected B cells. Furthermore, we have recently discovered that a BZLF1 promoter variant (known as Zp-V3) that is over-represented in certain EBV-positive malignancies (including NPC and AIDS-related lymphomas) relative to its frequency in non-malignant tissues confers enhanced lytic viral reactivation in vitro due to binding of the cellular NFAT transcription factor to the Zp-V3 variant (but not the prototype promoter, Zp-P). Although the Zp-V3 form of the promoter is relatively uncommon in type 1 EBV strains, it is present in all type 2 strains (which are very common in the malaria belt of Africa). These results suggest that enhanced lytic EBV infection increases the likelihood of EBV-induced lymphomas in vivo, and that a particular cancer-associated variant of the Z promoter promotes lytic infection in EBV-infected B cells. In this proposal, we will use two different humanized mouse models to compare the phenotypes of EBV containing the Zp-P form versus the cancer- associated (Zp-V3) form of the BZLF1 promoter, and to explore mechanism(s) by which lytic EBV infection promotes lymphomagenesis. We will also determine whether EBV loads are higher in malaria-infected children co-infected with Zp-V3 containing EBV strains versus Zp-P containing strains. Our Specific Aims are 1) to use humanized mouse models to examine the in vivo phenotypes of Zp-V3 versus Zp-P containing type 1 or type 2 EBV strains; 2) to compare the phenotypes of BALF5 (the viral DNA polymerase)-deleted versus BZLF1-deleted EBV mutants in humanized mice, and explore whether blocking lytic EBV DNA replication with the antiviral drug, acyclovir, inhibits the development of EBV-induced lymphomas; and 3) to determine whether the Zp-V3 promoter variant is associated with higher plasma levels of EBV in malaria-infected African children. We hypothesize that the EBV Zp-V3 variant will enhance EBV-induced lymphomas in humanized mice by increasing lytic EBV infection, and that this variant is also associated with enhanced lytic EBV replication in malaria-infected children. If so, these results will suggest that the presence of the Zp-V3 variant may be a useful biomarker for predicting increased risk of EBV-induced malignancy in humans.